Gene therapy has attracted wide attention as a method to treat various mammalian diseases and enhance production of specific proteins or other cellular products. Gene therapy is generally accomplished by introducing exogenous genetic material into a mammalian patient's cells. The introduced genetic material can be designed to replace an abnormal (defective) gene of the mammalian patient ("gene replacement therapy"), or can be designed for expression of the encoded protein or other therapeutic product without replacement of any defective gene ("gene augmentation"). Because many congenital and acquired medical disorders result from inadequate production of various gene products, gene therapy provides a means to treat these diseases through either transient or stable expression of exogenous nucleic acid encoding the therapeutic product.
Gene therapy can be accomplished by either direct transformation of target cells within the mammalian subject (in vivo gene therapy) or transformation of cells in vitro and subsequent implantation of the transformed cells into the mammalian subject (ex vivo gene therapy). In vivo gene therapy has been an area of particular interest, especially in transformation of somatic cells to repair particular defects in these cells, such as the administration of gene vectors to the upper respiratory tract in cystic fibrosis.
In addition to repair of somatic cells, in vivo gene therapy can also be used for systemic treatment, an area in which gene therapy has broad applications. Systemic treatment involves transfecting target cells with the DNA of interest, expressing the coded protein in that cell, and the capability of the transformed cell to subsequently secrete the manufactured protein into blood.
A variety of methods have been developed to accomplish in vivo transformation including mechanical means (e.g, direct injection of nucleic acid into target cells or particle bombardment), recombinant viruses, liposomes, and receptor-mediated endocytosis (RME) (for reviews, see Chang et al. 1994 Gastroenterol. 106:1076-84; Morsy et al. 1993 JAMA 270:2338-45; and Ledley 1992 J. Pediatr. Gastroenterol. Nutr. 14:328-37).
As with all therapies, the therapy that is most easily administered, least expensive, and most likely to realize patient compliance is the therapy of choice. Intestinal gene therapy provides such a therapy in the realm of gene therapy techniques. The intestinal epithelium is a particularly attractive site for in vivo gene therapy, largely due to the ease of access via an oral or other lumenal route. Administration of the exogenous nucleic acid to achieve in vivo transformation can be accomplished by non-invasive procedures. For example, the patient can simply take a pill composed of the exogenous nucleic acid or alternatively the exogenous nucleic acid formulation can be administered by some other non-invasive means (i.e., a means that does not require a major surgical procedure, such as endoscopic catheterization or rectal suppository incision).
Methods for accomplishing in vivo intestinal gene therapy have met with severe obstacles. Because the field has been primarily concerned with long-term gene therapy, most groups have shunned intestinal epithelial cells as targets for in vivo gene therapy due to the cells' rapid turn-over rate (2 to 4 days) (see, e.g., Sandberg et al. 1994 Hum. Gene Therap. 5:303-9). Efforts to achieve in vivo transformation may be further complicated by the mucus layer of the intestine, which is thought to block access of the gene therapy transforming formulation to the target cells (Sandberg et al., supra). The presence of high concentrations of DNAses in the intestinal tract is also thought to be a formidable barrier to the effective introduction of DNA into intestinal tract cells.
Many of the vectors and delivery systems developed for in vivo gene therapy either have their own inherent drawbacks or are not entirely suitable for in vivo intestinal gene therapy. For example, recombinant viruses, particularly retroviruses, may be slow in gaining FDA approval due to concerns generally associated with the administration of live viruses to humans. In addition, it has become clear that viral vectors present problems with the possibility of multiple administrations of the gene construct due to immune responses, and may greatly limit their utility. Mechanical means, such as the gene gun, are designed for use in transformation of skeletal muscle cells and are not particularly useful in intestinal cell transformation due to problems of access and to the delicate nature of organ.
Current methods of gene therapy designed to accomplish systemic therapeutic goals are limited to ex vivo techniques that require complex procedures to transform cells, pose the potential of rejection of the transplant, require at least minor invasive procedures, and limit implantation to modest numbers of cells. In vivo methods can also be used for systemic therapy, but these frequently require invasive procedures. Thus, there is a need in the field for a method to accomplish in vivo transformation of intestinal epithelial cells. The present invention addresses these problems by providing a non-invasive gene therapy method that uses naked nucleic acid constructs either alone or in combination with various adjuvants.